Transfer RNAs are transcribed as precursors with 5'and 3'end extensions which must be removed by processing. In the pre-tRNA maturation pathway, tRNase Z cleaves one nucleotide beyond the 3'end of the acceptor stem, endonucleolytically removing the 3'end trailer. A recognition and binding domain (RBD) is extruded from the body of tRNase Z remote from the active site. A co-crystal structure suggests that the globular head of the RBD principally binds to the elbow (D/T loops) of tRNA, far from the scissile bond. We propose to investigate the RBD of tRNase Z by posing three questions: (1) How does each residue in the head of the RBD contribute to substrate binding and catalysis? (2) Is the RBD an independently folding unit? (3) What are the contacts between the RBD and pre-tRNA? This investigation will shed light on a novel mechanism for substrate recognition. Transfer RNA (tRNA) is central to the process of protein synthesis. Mutations in tRNAs are associated with maternally transmitted mitochondrial diseases and syndromes. Additionally, the gene that encodes tRNase Z, an enzyme in the tRNA maturation pathway, has been associated with an elevated risk of prostate cancer, making the proposed research biomedically relevant.